In vivo, cells are maintained in three-dimensional microenvironments regulated by physical, chemical and biological factors. The microenvironments are characterized by (1) a short distance between cells, (2) a continuous nutrient supply and waste removal, (3) a common set-point temperature, and (4) a minimal stress level. On the other hand, conventional cell culture systems in which cells are grown in monolayers in flasks or in dishes substitute only imperfectly for such microenvironments. For example, convection, or bulk fluid movement, is increased and can bring about undesired variation in temperature, solute concentration, dissolved gas concentration and can lead to surface tension differences at a gas-solution interface (i.e., Marangoni effect). Such variations cause mixing, as opposed to diffusion, of molecules secreted by cells in the culture. Consequently, secreted molecules rapidly distribute over the entire volume of the culture, interfering with both autocrine and paracrine signaling that is associated in vivo with cell-cell interactions. Additionally, three-dimensional cell growth does not take place in conventional cell culture environments, as cell-to-cell contact is largely absent.
In the past decade, research has shown that factors such as surface tension, energy dissipation and associated convective flow, and fluidic resistance affect the behavior of fluids in conventional cell culture, whereas these factors can be attenuated by providing suitable microenvironments. The field of microfluidics studies how these behaviors affect culture systems, and how they can be worked around or exploited for new uses, particularly for cell culture systems having a size scale that provides environments for cells that are more similar to their native in vivo culture environments than those of conventional culture systems.
Microfluidic systems can be restrict flow such that small molecules move in the culture only via diffusion (i.e., essentially convection-free). Because microenvironments are so small, gas-liquid interfaces can be eliminated. As such, convection-free in vitro microenvironments offer opportunities to study cellular processes such as autocrine and paracrine functions that cannot readily be studied in conventional systems. A related advantage of microfluidics is that microenvironments can enhance desirable cell-to-cell contact.
As culture systems are scaled down, factors such as diffusion, surface tension and viscosity become important. The skilled artisan is familiar with such factors, which are summarized, e.g., in Atencia J & Beebe D, “Controlled microfluidic interfaces,” Nature 437:648-655 (2005); Walker G, et al., “Microenvironment design considerations for cellular scale studies,” Lab. Chip. 4:91-97 (2004); Yu H, et al., “Diffusion dependent cell behavior in microenvironments,” Lab. Chip. 5:1089-1095 (2005); and U.S. Published Patent Application No. 2004/0259177, each of which is incorporated herein by reference as if set forth in its entirety.
Although embryonic stem cells have been cultured and monitored in microenvironmental systems, long-term study of cell proliferation is largely unexplored. Isolation and in vitro culturing of adult stem cells remain unsatisfactory. Rizvi A & Wong M, “Epithelial stem cells and their niche: there's no place like home,” Stem Cells 23:150-165 (2005), incorporated herein by reference as if set forth in its entirety. Current techniques for isolating adult stem cells include identifying cell surface markers and functional studies (e.g., dye efflux and patch clamping), whereas current techniques for culturing adult stem cells utilize macroenvironments.
Likewise, isolation and in vitro culturing of many primary cells, such heterogeneous primary epithelial cells, remain unsatisfactory. In fact, most primary cells die when transferred to primary culture. Any cell lines that emerge from such culture typically reflect a small subpopulation with a low frequency of mutation that permits genetic adaptation to non-physiological culture conditions. Consequently, karyotypes of the resulting cell lines are so abnormal that such cells would not survive in vivo when used for transplant.
For the foregoing reasons, there is a need for microfluidics methods and systems for evaluating co-cultures of heterogeneous cells, as well as the factors that influence proliferation.